Method for stabilization of proteins in solution

ABSTRACT

The present invention relates to a method for stabilization of analytes in solutions of solubilized body samples. The method comprises the steps of solubilizing the body samples obtained from a subject in a suitable sample medium and stabilizing said body sample contained within the sample medium by heating said sample medium for a certain period of time. A further aspect of the invention is a method of a denaturing immunoassay of proteins in solution. The method comprises the steps of bringing a sample containing proteins into contact with a denaturing agent and heating said sample in the presence of the denaturing agent to allow the protein to be denatured.

This application is a continuation of U.S. patent application Ser. No.11/701,522, filed Jan. 31, 2007; which claims foreign priority under119(a)-(d) of EP Application No. 06101298.5, filed Feb. 3, 2006. Theabove applications are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method for stabilization of analytesin solutions of solubilized body samples. The method comprises the stepsof solubilizing the body samples obtained from a subject in a suitablesample medium and stabilizing said body sample contained within thesample medium by heating said sample medium for a certain period oftime. A further aspect of the invention is a method of a denaturingimmunoassay of proteins in solution. The method comprises the steps ofbringing a sample containing proteins into contact with a denaturingagent and heating said sample in the presence of the denaturing agent toallow the protein to be denatured.

BACKGROUND OF THE INVENTION

In medical diagnosis, one crucial step influencing the results of adiagnostic method applied is the storage and transportation of thesample. This is especially due to the fact that the analytes that are tobe detected within a body sample may be subject to degradation or mayotherwise be influenced during the transport and storage. In order toensure accurate determination of diagnostically relevant analytes theintegrity of such analytes within the samples must be maintained untilanalysis is performed.

In the art, one approach to overcome stability problems for analytesseeks to avoid any transportation and storage of samples by implementingpoint of care testing systems that allow for analysis of body samplesdirectly at the site of patient care, where the sample has beenobtained. However, such approaches are limited respective the analyticaltechnologies that may be applied. For complex analytical methodsrequiring laboratory equipment, ways to enable transportation andstorage of samples without interfering with the integrity of analytesmust be found.

One way to allow for transportation of body samples is to preserve thecells contained within the samples and thus to enable for cell basedcytological or histological examination of the sample material. Examplesfor preservation solutions for cytological examinations compriseDigene's Universal Collection Medium (WO9931273), Cytyc®'s PreservCyt®Solution (EP0511430), or Surepath® Cytorich® solution. These media aredesigned to preserve cellular morphology and also the integrity ofcellular proteins to allow for cytological examination of the preservedcellular samples. All such preservation solutions comprise alcohols asfixatives. Generally spoken, preservation of the analytes in suchprocedures is achieved by addition of chemical preservative substances.In cases where the cellular integrity is no longer needed for thediagnostic procedure the integrity of the analytes in a solution maysimilarly be achieved by addition of chemical preservative substances.The main disadvantage of this approach is that preservatives are oftentoxic substances that may harm operators and may damage the environmentin case of spill. Additionally the requirements for waste disposal aswell as the cost for waste disposal are increased when using chemicaladditives for sample stabilization.

One further approach to solve the problem of stability of analytes inbody samples is refrigeration or freezing of the body samples duringtransportation and storage. It is known that biological substances maybe preserved by refrigeration for a certain period of time and thatfreezing of biological material below −20° C. may be applied forpreservation of long term storage. This solution however has thedisadvantage of energy consumption and posing hurdles on thetransportation process. It cannot always be ensured that refrigerationor maintaining temperatures below ±20° C. may be upheld throughout thetransportation. This turns out to be a serious problem in cases wherethe stability of an analyte is not proven under certain temperatureconditions. Therefore the method of the present invention involving aheating step provides an easy way to stabilize samples fortransportation and storage without the need for addition ofpreservatives or the need for refrigeration.

Additionally, the heating step if performed in a medium that comprisesdenaturing agents may contribute to the reproducibility and accuracy ofsubsequent analytical steps. If the heating step is e.g. performed inthe presence of Sodium Dodecyl Sulfate that interacts with proteins inthe respective sample, a denatured structure of the proteins isobtained. This denatured structure may in certain cases be of advantagefor the reproducible and quantitative determination of the protein insolution.

In the art, methods for denaturing protein bio-assays are used in thecase of the Laemmli-System in denaturing Polyacrylamide GelElectrophoresis in the presence of SDS (known as SDS PAGE). In this casethe samples containing proteins are also heated in the presence of SDSto allow denaturation of the protein and are subsequently separatedaccording to their size in an electrophoretic step. The purpose of thedenaturation in this method is denaturation of the protein to allow foran overall linearization of the proteins together with homogeneousloading of the proteins with SDS ions. This ensures that proteins incontrast to their native form exhibit an electrical charge that isproportional to the overall size of the molecules. The overall methodallows for separation of the molecules by size.

The present invention is based on the inventor's findings that analytesin solubilzed body samples may be stabilized by heating of the samplesolution for a certain period of time. According to the presentinvention heating may for example be performed in a suitable samplemedium. The method according to the present invention overcomes thedrawbacks of the methods known in the art. The method found by theinventors does not make use of chemical preservatives that may causeharm to animals, human beings or the environment and does not impose theneed for refrigeration or keeping up of other energy consumingconditions during the whole process of transportation and storage.

The effect of denaturing of the proteins during the heating step allowsfor accurate determination of the proteins within the samples usingdenaturing bioassays. In contrast to the methods known in the art, thedenaturing immunoassay is immunochemical detection directly from thesample solution employing a solid phase fixed detection probe thatspecifically binds to the proteins within the sample. An electrophoreticstep is not necessarily comprised in the overall method. The purpose andeffect of the denaturation in the context of this method is thereforenot due to the generation of homogeneous size dependant electric chargeof the proteins but is in part due to the linearization and therefore toimproved accessibility of antigenic epitopes within the proteins.

SUMMARY OF THE INVENTION

The present invention is based on the inventors findings illustrated inthe following description of the invention and exemplified in theExamples provided herein that analytes in solutions of body samples maybe stabilized by a heating step. The heating step comprises the heatingof a sample solution preferably in the presence of a denaturing agentfor a period of time to a temperature sufficient to allow the sample tobe stabilized. The invention furthermore pertains to the improvement ofaccurate and quantitative or semi-quantitative determination of e.g.protein-analytes in solutions by denaturing the protein containedtherein by heating in the presence of a denaturing agent to allowpartial denaturation of the protein within the solution and subsequentimmuno-chemical quantitative or semi-quantitative determination of theprotein. The method is e.g. applicable to preservation of analytes forsubsequent assay in the course of assessment or support of diagnosis onthe basis analyte levels in said body samples. The method may be appliedto any kind of body sample obtained for diagnostic or whatsoeverpurpose.

One aspect of the present invention is a method for stabilization ofanalytes in solutions of solubilized body samples. The method comprisesthe steps of solubilizing the samples material obtained from a subjectin a suitable sample medium and stabilizing the sample contained withinthe sample medium by heating said sample medium for a certain period oftime. The method is suited for stabilization of analytes and mayespecially be applied under circumstances where cooling or refrigerationis not possible and where chemical preservative substances shall or mustbe avoided.

A second aspect of the present invention is a method for denaturingimmunoassay of proteins in solution, wherein at least one step of theimmunoassay is carried out in presence of the denaturing agent in aconcentration that allows to uphold the denatured status of an analyteprotein.

A third aspect of the present invention is a sample comprising analytessuited for biochemical or immunochemical analysis to be used e.g. as astandard or control sample for an assay or assay kit, that has beentreated according to the method of the present invention.

A fourth aspect of the present invention is a device for heating samplesaccording to a method of the present invention, the device beingcharacterized in having a timer and drillings suited to fit the samplecollection vials.

A fifth aspect of the present invention is a sample collection vialcontaining a medium comprising agents suited for stabilization and/ordenaturation of body samples according to a method of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stability of p16^(INK4a) in solubilized samples. Thelevels of p16^(INK4a) were determined in samples from the human cervixuteri using an ELISA. The cervical samples were solubilized in a samplemedium and thereafter incubated for several days at ambient temperature.Aliquots from each sample were taken for determination of thep16^(INK4a) levels on day 1 (immediately after heat treatment), day 2,day 3 and day 7. The graph shows p16^(INK4a) levels determined in 7samples at different points in time and thus displays instability ofp16^(INK4a) within the particular patient samples under the givenconditions. For details see Example 1.

FIG. 2 shows the stability of p16^(INK4a) in solubilized samples afterheat treatment. The levels of p16^(INK4a) were determined in samplesfrom the human cervix uteri using an ELISA. The cervical samples weresolubilized in a sample medium, heat treated in a water bath for 15 minat 95° C. and thereafter incubated for several days at ambienttemperature. Aliquots from each sample were taken on day 1 (immediatelyafter heat treatment), day 2, day 5 and day 8. The graph showsp16^(INK4a) levels determined in 11 different patient samples anddisplays stability of p16^(INK4a) upon heat treatment. For details seeExample 1.

FIG. 3 shows the impact of the duration of the heat treatment onstability of p16^(INK4a) in solubilized samples after heat treatment.The levels of p16^(INK4a) were determined in samples from the humancervix uteri using an ELISA. 14 different cervical samples wereincubated up to 45 min above 95° C. 4 consecutive aliquots were takenafter 15 min, 25 min, 35 min and 45 min from start of heat treatment.The p16^(INK4a) concentration was determined in duplicate from eachaliquot separately, using the p16^(INK4a) ELISA. Average concentration,including error bars of 4 determinations, as well as coefficient ofvariation between 4 determinations are shown. For details see Example 1.

FIG. 4 shows the impact of the temperature applied during heat treatmenton stability of p16^(INK4a) in solubilized samples after heat treatment.The levels of p16^(INK4a) were determined in samples from human cervixuteri using an ELISA. A mixed pool of several patient samples positivefor p16^(INK4a) was divided in 4 aliquots which were either leftuntreated or incubated for 10 min at 80° C., 90° C., 99° C.,respectively. Aliquots were taken on day 1 (immediately after heattreatment), day 3, day 6 and day 8. For details see Example 1.

FIG. 5 shows the stability of gCatenin in six solubilized samples. Thelevels of gCatenin were determined in samples from the human cervixuteri using an ELISA. 3 Cervical samples were solubilized in samplemedium immediately after the samples had been obtained and wereincubated thereafter for several days at ambient temperature. Aliquotswere taken on day 1 (immediately after heat treatment), day 2, day 5 andday 6. The levels of gCatenin determined in each sample using an ELISAare graphically represented. For details see Example 1.

FIG. 6 shows the stability of gCatenin in seven solubilized samplesafter heat treatment. The levels of gCatenin were determined in samplesfrom human cervix uteri using an ELISA. Seven Cervical samples weresolubilized in sample medium immediately after the samples had beenobtained, heat treated in a water bath for 15 min at 95° C. and wereincubated thereafter for several days at ambient temperature. Aliquotswere taken on day 1 (immediately after heat treatment), day 2, day 4,day 8, day 10 and day 15. For details see Example 1.

FIG. 7 shows the stability of Ep-CAM in seven solubilized samples. Thelevels of Ep-CAM were determined in samples from human cervix uteriusing an ELISA. Six Cervical samples were solubilized in sample mediumimmediately after the samples had been obtained and were incubatedthereafter for several days at ambient temperature. Aliquots were takenon day 1 (immediately after heat treatment), day 3, day 5 and day 7. Fordetails see Example 1.

FIG. 8 shows the stability of Ep-CAM in seven solubilized samples afterheat treatment. The levels of Ep-CAM were determined in samples fromhuman cervix uteri using an ELISA. 7 Cervical samples were solubilizedin sample medium immediately after the samples had been obtained, heattreated in a waterbath for 15 min at 95° C. and were incubatedthereafter for several days at ambient temperature. Aliquots were takenon day 1 (immediately after heat treatment), day 2, day 5 and day 6. Fordetails see Example 1.

FIG. 9 shows the p16^(INK4a) ELISA results in the presence of 0.5% SDSand 3% TRITON® X100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethyleneglycol solution). The p16^(INK4a) levels were measured in an ELISA outof samples containing 3% TRITON® X100 and 0.5% SDS. Samples withcytologically reported abnormality of the epithelium of the uterinecervix were applied. The graph shows the OD measured in the ELISA incorrelation to the cytological diagnosis available. For details seeExample 1a.

FIG. 10 shows the p16^(INK4a) levels determined in patient samples byELISA and LIA; both performed in the presence of 0.3% SDS. Thep16^(INK4a) levels were determined in samples from human cervix uteriusing ELISA and LIA immunoassays. Immunoassay was performed in thepresence of 0.3% SDS for both assay types. 12 Cervical samples weresolubilized in sample medium immediately comprising 0.3% SDS. Sampleswithin the sample medium were directly applied to the immunoassay. Bothtypes of immunoassay prove to detect p16^(INK4a) protein levels in thepresence of elevated concentration of SDS. For details see Example 2.

FIG. 11 shows the p16^(INK4a) levels determined in patient samples byLateral Flow assay performed in the presence of 0.3% SDS. Tablerepresenting data were obtained for Cytology and Histology specimensfrom patients in correlation to the representation of the resultsobtained when applying the sample solution comprising 0.3% SDS and 1%TRITON® X100 directly to the Lateral flow dipstick. It could be seenthat in the dipstick assay format p16^(INK4a) as correspond to severeddysplastic lesions in patients may be detected by generation of aspecific band. This demonstrates that the dipstick format may be appliedaccording to the present invention in the presence of denaturing agents.For details see Example 3.

DETAILED DESCRIPTION OF THE INVENTION

Based on the findings of the inventors stabilization of analytes in bodysamples may be achieved by solubilizing said body samples in a suitablesample medium and subsequently subjecting the solubilized samples to aheating step before transportation and storage of the samples.Furthermore the step of heating of the samples in the presence ofdenaturing agents may contribute to the accurate quantitativedetermination of analytes in the samples.

Medical examination procedures are commonly accompanied or supported bythe assay of analytes within body samples. Such analytes may e.g. beassayed in clinical chemistry laboratory procedures.

The expression analyte as used in the context of the present inventionshall refer to molecules that may be detected in the course of ananalytical testing procedure. In certain embodiments of the presentinvention the analytes are e.g. nucleic acid as well as (poly)peptidemolecules. Such analytes thus comprise e.g. RNA (mRNA, hnRNA, etc.), DNA(cDNA, genomic DNA, etc.), proteins, polypeptides, proteoglycans,glycoproteins and the respective fragments of these molecules.

Analytes may be whatsoever molecules. In certain embodiments of thepresent invention analytes are molecules with a special relevance forthe detection of diseases in mammalians. Diseases according to thepresent invention may comprise any kind of medically relevant conditionscomprising but not limited to neoplastic, inflammatory, infectious,degenerative, genetic, proliferative and vascular diseases as well aspremalignant and malignant cancerous conditions. In certain embodimentsof the invention premalignant and malignant cancerous conditions maycomprise neoplastic disorders such as tumors. Tumors may comprise tumorsof the head and the neck, tumors of the respiratory tract, tumors of theanogenital tract, tumors of the gastrointestinal tract, tumors of theurinary system, tumors of the reproductive system, tumors of theendocrine system, tumors of the central and peripheral nervous system,tumors of the skin and its appendages, tumors of the soft tissues andbones, tumors of the lymphopoietic and hematopoietic system, etc. Tumorsmay comprise for example neoplasms such as benign and malignant tumors,carcinomas, sarcomas, leukemias, lymphomas or dysplasias. In aparticular embodiment, the tumor is for example cancer of the head andthe neck, cancer of the respiratory tract, cancer of the anogenitaltract, cancer of the gastrointestinal tract, cancer of the skin and itsappendages, cancer of the central and peripheral nervous system, cancerof the urinary system, cancer of the reproductive system, cancer of theendocrine system, cancer of the soft tissues and bone, cancer of thehematopoietic and lymphopoietic system.

Tumors of the anogenital tract may comprise cancer of the perineal andthe scrotal skin, cervical cancer, cancer of the vulva, cancer of thevagina, cancer of the penis, cancer of the anus, etc. Cervical cancermay comprise squamous lesions, glandular lesions or other epithelialtumors. Squamous lesions comprise, e.g., cervical intraepithelialneoplasias (mild, moderate and severe dysplasia), carcinoma in-situ,squamous cell carcinoma (e.g., keratinizing, nonkeratinizing, verrucous,warty, papillary, lymphoepithelioma-like). Glandular lesions maycomprise atypical hyperplasias, adenocarcinoma in-situ, adenocarcinoma(such as, e.g., mucinous, endometrioid, clear cell, adenoma malignum,papillary, serous or mesonephric adenocarcinoma). Other epithelialtumors may comprise adenosquamous carcinoma, glassy cell carcinoma,adenoid cystic carcinoma, adenoid basal carcinoma, carcinoid tumor,small cell carcinoma and undifferentiated carcinoma. For more detailedinformation, confer “Kurman, R., Norris, H., et al., Tumors of theCervix, Vagina, and Vulva, Atlas of Tumor Pathology, 1992, AFIP,” thecontents of which shall be incorporated herein by reference.

Gastrointestinal tumors may comprise colon cancer, cancer of the colonascendens, of the colon descendens, of the colon transversum, of thesigmoidum, of the rectum, cancer of the small intestine, cancer of thejejunum, cancer of the duodenum, gastric cancer, oesophageal cancer,liver cancer, cancer of the bile, cancer of the biliary system,pancreatic cancer, etc. A comprehensive overview over gastrointestinallesions is given in “Hamilton Sr, Aaltonen L A (Eds.): World HealthOrganization Classification of Tumours, Pathology and Genetics of Tumorsof the Digestive System, IARC Press: Lyon 2000,” which shall beincorporated herein by reference.

Tumors of the respiratory tract may comprise any malignant condition ofthe respiratory tract such as, e.g., cancer of the lung, the alveoles,the bronchioles, the bronchial tree and the broncus, the nasopharyngealspace, the oral cavity, the pharynx, the nasal cavity and the paranasalsinus. Lung cancer such as small cell lung cancer, non-small cell lungcancer, squamous cell lung carcinoma, small cell lung carcinoma,adenocarcinoma of the lung, large cell lung carcinoma, adeno-squamouslung carcinoma, carcinoid tumor of the lung, bronchial gland tumor or(malignant) mesothelioma. An overview over tumors of the respiratorytract may be found in Colby T V, et al.: Tumors of the Lower RespiratoryTract, Atlas of Tumor Pathology, Third Series, Fascicle 13, AFIP:Washington 1995,” which shall be incorporated herein by reference.

Tumors of the urinary system may comprise bladder cancer, cancer of thekidney, renal pelvis, cancer of the ureters and cancer of the urethra,etc. Tumors of the reproductive system may comprise cancer andprecursory stages thereof of the ovary, the uterus, the testis, theprostate, the epididymis, etc.

Molecules with a special relevance for disease that may be used asanalytes according to the present invention may be molecules that may beused for detection of the presence or absence of such disease. Themolecules may be indicative of the presence or absence of the disease invarious ways. Such ways may comprise altered expression levels (e.g.increased, decreased, temporally or locally altered) as well asexpression of molecules with altered characteristics. Such alteredcharacteristics may comprise any kind of modified molecules. Suchmodifications comprise modifications of the sequence of molecules (suchas mutations, deletions insertions, etc.) as well as any other type ofmodifications of the molecules (such as e.g. chemical modifications,phosphorylations, glycosylations, derivatization, dephosphorylationetc.).

In certain embodiments of the present invention the analytes may e.g. becancer marker molecules or tumor markers. The analytes may be selectedfrom a group comprising markers for cell proliferation, markerscharacteristic for apoptosis, markers for cell surface epitopes, markersassociated with viral infection or viral activity in cells (e.g. markersfor infection by high risk human papilloma virus selected from a groupcomprising HPV16, HPV18, HPV31, HPV 33, HPV35, HPV 39, HPV 45, HPV 51,HPV 52, HPV56, HPV 58, HPV 59, HPV 66 and HPV 68), markerscharacteristic for cell differentiation etc. In certain preferredembodiments the analytes are cyclin dependent kinase inhibitors.

Analytes may e.g. comprise molecules derived from proteins selected froma group comprising p13.5, p14, p15, p16 (also referred to p16^(INK4a)),p19, p21, p27, p53, pRb, p14ARF, cyclin A, cyclin B, cyclin E, MDM-2,CDC2, Id1, osteopontine, GRP, renal dipeptidase, her2/neu, TGFβIIreceptor, Cytokeratines, (e.g. cytokeratin 8, cytokeratin 10,cytokeratin 18), mucin antigens (MUC1, MUC2, Tn, STn), EpCAM andgCatenin, concanavalin A receptor, GalNacTransferase,oligosaccharyltransferase, lectins (ConA, WGA, PNA, UEA I, DBA, SBA,SNA), plakophilin, vimentin, CD antigens (e.g. CD3, CD16, CD18, CD4,CD8, CD56, CD19, CD20), HPV associated markers e.g. derived from HPVgenes L1, L2, E1, E2, E4, E5, E6 or E7, CDC6, MCM2, MCM3, MCM4, MCM5,MCM6, MCM7, CDC7 protein kinase, Dbf4, CDC14 protein phosphatase, CDC45and MCM10, Ki67, Ki-S2, PCNA, a helicase, a topoisomerase, Topo2alpha,transcription factors, members of the E2F-family, Brn-3a, Brn-3b or POLDetc.

Analytes may be molecules that are detected on a qualitative,semiquantitative as well as a quantitative basis. A quantitative valuemay e.g. be represented in terms of a concentration. A semiquantitativevalue may be expressed in terms of a scale of levels e.g. undetectablelevels, low levels, intermediate levels, high levels or any othersuitable mode. The level of an analyte may also be represented in termsof a dependent parameter such as the intensity of a signal generated inan assay format in response to the presence of a marker molecule. Forthe expression of the signal intensity in semi-quantitative as well asin quantitative assays relative units may be used for the expression ofthe determined amounts of analyte. The relative units used may bedenominated in any suitable manner such as e.g. as units per mL ofsample (U/mL). Such units may be directly related to real molarconcentration of analytes as measured in a solution but may also bearbitrary units as applicable for the respective assay format.

Analytes are detected by immunoassay according to the present invention.Immunoassay according to the present invention is any immunochemicaldetection of an analyte. Such immunochemical detection may in certainembodiments comprise assays where one or more probes or antibodies areused for capture of the analyte molecules and subsequent detection ofthe analyte probe/antibody complex. Such reaction may either beperformed in solution or fixed to a solid phase. Those of skill in theart know appropriate immunoassays. Such assay may comprise ELISA andvarious kind of other immunoassays. Such immunoassays may comprise butnot be limited to for example immunoprecipitation or immunologicalassays, such as EIA, ELISA, RIA, ECLIA, LIA, lateral flow assays, flowthrough assays, immunochromatographic strips, etc. Immunoassays for usein the invention may comprise competitive as well as non-competitiveimmunoassays. In certain embodiments antibodies or probes fixed to solidphases may be employed in the immunoassays. Solid phases may comprisevarious embodiments of solid substances such as planar surfaces,particles (including micro-, nano-particles or even smaller particles).In certain embodiments, particles may be provided as spheres, beads,colloids, or the like.

The fixation of reagents to the solid phase in a test kit or an in-vitrodiagnostic device may be carried out via direct fixation or via indirectfixation. Direct fixation may be carried out by covalent binding,non-covalent binding, association, or adsorption to surfaces. Indirectfixation may be carried out through binding of the antibody to agentswhich themselves are directly fixed to solid phases. Binding agentsinclude avidin, streptavidin, biotin, digoxigenin, antibodies or thelike.

The immunoassay may comprise one or more further reactions withdetecting agents either recognizing the analytes or preferablyrecognizing probes used to recognize the analytes. The detectionreaction further may comprise a reporter reaction indicating the levelof the analytes.

Detection systems may be e.g. chromogenic systems, luminescence systems(electroluminescence, bioluminescence, photoluminescence,radioluminescence, chemiluminescence, electrochemiluminescence),fluorescence based systems, conductivity based detection systems,radiation (light, UV, X-ray, gamma etc.), plasmon resonance (e.g.Surface Plasmon Resonance SPR) or any other known method.

According to the present invention the body sample solubilized withinthe sample medium may directly be applied as specimen for theperformance of the immunoassay. In certain embodiments of the presentinvention high concentrations of detergents may be present duringimmunoassay. The immunoassay may therefore in certain embodiments of thepresent invention be an immunoassay that is especially tolerant againstdenaturing conditions. In certain embodiments of the invention theimmunoassays are tolerant against concentrations of SDS above 0.1%, in apreferred embodiment the immunoassays are tolerant against SDSconcentrations above 0.3%. In another preferred embodiment theimmunoassays are tolerant against concentrations of SDS above 1%.

The detection of analytes is commonly performed using one or more“probes” (used interchangeably with the term “binding agents” within thepresent invention) for the detection of the analyte. The probe may forexample be an antibody specifically binding to the analyte. The term“antibody” in all its grammatical forms shall in the context of thepresent invention refer generally to antigen binding molecules includingbut not limited to monoclonal and polyclonal antibodies, fragments ofantibodies, antigen binding epitopes, mini-antibodies, peptidomimeticswith antigen-binding properties, anticalines and diabodies. Generallythe detection of the analyte shall comprise detection of the presence orabsence and or the level of analytes.

The expression “body sample” as used herein comprises any body samplesof any kind and nature. Examples of such body samples are secretions,swabs, lavages, body fluids, semen, cell- and tissue-samples, blood,smears, sputum, urine, stool, liquor cerebrospinalis (short denominatedas “liquor” herein), bile, gastrointestinal secretions, lymph, bonemarrow, aspirates and biopsies of organs such as needle or punchbiopsies and (fine)-needle aspirates. In particular, smears, swabs andbiopsies are indicated when the detection of anogenital cancers, e.g.cervical cancers, is concerned. The term biopsies as used throughoutthis text shall comprise all kind of biopsies known to those of skill inthe art. Thus biopsies as used in the context of the present inventionmay comprise e.g. resection samples of tumors, tissue samples preparedby endoscopic means or punch- or needle-biopsies of organs. Biopsiescomprises specimens obtained by several different methods such as coldknife biopsies, LEEP (loop electrocautery excisional procedure)biopsies, etc. Body samples according to the present invention may incertain embodiments of the invention refer to specimens comprisingcellular material obtained from the human body. Such samples may e.g.comprise specimens that are suited for use in cytological examination.Examples are swab samples, liquid based cytology samples and smears.

Liquid based cytology samples may comprise any kind of liquid basedcytology sample comprising e.g. cell or tissue samples preserved in anystandard sample collection, storage or transportation medium, known tothose of skill in the art such as e.g. commercially availablepreservation media (formalin solution, Cytyc “PreservCyt” or “CytoLyt”,Digene “Universal Collection Medium”, Tripath Imaging “Cytorich”, etc.).Alternatively cell preservation media for liquid based cytology samplesmay contain a mixture of one or more selected from a group comprisingalcohols, aldehydes, ketones, acids, metal-ions or sublimates, ethersetc. for preservation of cellular components.

Alcohols include methanol, ethanol, (n- or i-) propanol, (n-, i- or t-)butanol or higher branched or unbranched alcohols. Aldehydes includeformaldehyde, acetaldehyde, glutaraldehyde, etc. Ketones such as Acetonemay be used. Acids for use in standard sample media include organicacids (acetic acid, trichloro-acetic acid, salicylic acid, picric acid)or inorganic acids such as e.g. chromic acid. Standard sample solutionsmay comprise metals such as silver, copper, chromium, mercury, osmium,uranium. Solutions of salts such as uranyl-acetate, potassiumbichromate,ammonium sulfate, etc. may be components of preservative media. Inpreferred embodiments the Liquid based cytology specimens are especiallysuited for subsequent immunochemical detection of protein analytes inthe samples.

In certain embodiments of the present invention the body sample may beobtained from a device that is used for obtaining a specimen for testingin another procedure before such device is discarded. Devices may forexample comprise any kind of brush, broom, spatula, swab, etc. Suchdevices comprise e.g. swabs made from Dacron®, cotton or any othersuitable fiber, brushes such as Endocervical brush, Medscand system forcervical sampling, CERVEXBRUSH®, CERVEXBRUSH COMBI™ from Rovers MedicalSystems, N.V., CYTOBRUSH® from Medscand Medical AB and Digene Corp.'sCERVICAL CELL SAMPLER™.

In certain embodiments the body sample according to the presentinvention is obtained from the devices after sample material for anothertesting procedure has been obtained. For example the sampling device maybe used for preparation of a conventional smear sample and maythereafter be contacted to a sample medium according to the presentinvention. Alternatively the sampling device may also be used forpreparation of any kind of liquid based cytology sample and be contactedto the sample medium according to the present invention subsequently. Inthe case of liquid based cytology samples it is also intended by theinventors that part of the liquid based cytology sample may be used tobe contacted with the sample medium and be solubilized for stabilizationor analysis by a denaturing immunoassay.

Contacting of the sampling device to a sampling medium may compriseimmersing the sampling device into a vial containing the sample medium.In certain embodiments the sampling device is immersed in the medium tofully cover the part of the sampling device that has been contacted tothe human body with the sample medium. Upon immersing the samplingdevice into the sample medium the device may be swirled in the medium.Alternatively the medium together with sampling device may be shaken orvortexed. In certain cases the sampling device may be contacted to thesample medium in a way that the device or part of the device is storedor left within the sample medium for a period of time to allow completedissolution of the cells or cell debris. In certain cased the samplingdevice may e.g. be heated together within the sample medium.

Generally the performance of an immunoassay may take place immediatelyafter the body sample has been contacted with the sample medium or maytake place after a certain period of time. In certain embodiments of theinvention the immunoassay is performed directly when the lysis of thebody sample is completed or is believed to be completed. In certainfurther embodiments of the invention after completion of the lysis ofthe body sample the sample solution is stored for a period of time thatmay range between minutes up to weeks. In one preferred embodiment thetime between lysis of the body sample in the sample medium and theperformance of the immunoassay is a period of time not shorter than 1Minute and not longer than 90 days, in an even more preferred embodimentthe period of time is not shorter than 1 hour and not longer than 14days and in another preferred embodiment the period of time is notlonger than 7 days. In certain embodiments the time period may even beup to the range of years.

Body samples according to the present invention may be contacted to asample medium immediately after obtaining the samples or even after acertain span of time has passed by. During this time the body samplesmay e.g. be refrigerated, frozen or preserved in a cell preservativesolution. The methods for such cell preservation or for preservation byfreezing or refrigeration are known to those of skill in the art.

According to the present invention one body sample may be used for thedetection of one or more analytes in immunochemical analysis. Thedetection of the analytes may be performed in one single assay or inmultiple distinct assays. In certain embodiments of the inventionaccordingly only an aliquot of a total body sample is employed for thedetermination of a single analyte out of the body sample.

The terms “solubilized” or “solubilization” as used in the context ofthe present invention shall mean that the components of a body sampleare contacted to a sample medium and are at least in part dissolved inthe sample medium and subsequently the components of the body sample areat least in part represented by the components of the solution.

The term “sample medium” as used in the context of the present inventionmay be any liquid known to those of skill in the art to be suited forsolubilization of cellular components or of whole cells. The samplemedium may, for example, be organic or aqueous solutions of chaotropicagents such as e.g. urea, GuaSCN, formamide, of detergents such asanionic detergents (e.g. SDS, N-lauryl sarcosine, sodium deoxycholate,alkyl-aryl sulfonates, long chain (fatty) alcohol sulfates, olefinesulfates and sulfonates, alpha olefine sulfates and sulfonates, sulfatedmonoglycerides, sulfated ethers, sulphosuccinates, alkane sulfonates,phosphate esters, alkyl isethionates, sucrose esters), cationicdetergents (e.g. cetyl trimethylammonium chloride), non-ionic detergents(e.g. TWEEN® 20 (polyoxyethylene-sorbitan monolaurate), Nonidet P-40(nonylphenylpolyethylene glycol), TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenylpolyethylene glycol solution), IgepalCA-630 (octylphenoxy)polyethoxyethanol), N-octyl-Glucosid) or amphotericdetergents (e.g CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate),3-dodecyl-dimethylammonio-propane-1-sulfonate, lauryldimethylamineoxide) and/or of alkali hydroxides such as e.g. NaOH or KOH. In certainembodiments the sample medium may also comprise inorganic or organicacids as components such as formic acid, acetic acid, phosphoric acidetc. In certain embodiments, where lysis of cells may be achievedwithout the use of detergents, hyper- or hypotonic solutions or buffersor simply water or an organic liquid may be used as solvent. Any liquidthat is suited to solubilize the cellular components of body samples intotal or in parts may be regarded as a sample medium as used herein.Thus sample media as used herein may but need not contain buffersubstances or have buffer capacity.

Generally any suitable liquid may be used in the sample medium of thepresent invention. The liquid may be organic or inorganic and may be apure liquid, a mixture of liquids or a solution of substances in theliquid and may contain additional substances to enhance the propertiesof the solvent. In certain embodiments, where lysis of cells may beachieved without the use of detergents, hyper- or hypotonic solutions orbuffers or simply water or an organic liquid may be used as solvent. Anyliquid, that is suited to solubilize the cellular components of bodysamples in total or in parts may be regarded as a sample medium as usedherein. Thus sample media as used herein need not contain buffersubstances or have buffer capacity. However in certain embodiments ofthe invention the sample media may have buffer capacity and may containbuffer substances.

In one embodiment, the sample medium is designed, so that cells, celldebris, nucleic acids, polypeptides, lipids and other biomoleculespotentially present in the raw sample are solubilized. In furtherembodiments of the present invention, the solvent may be designed toassure differential solubilization of specific components of the bodysample, leaving other components unsolubilized.

The sample medium for solubilizing the body sample according to thepresent invention may furthermore comprise one or more agents thatprevent the degradation of components within the raw samples. Suchcomponents may for example comprise enzyme inhibitors such as proteinaseinhibitors, RNAse inhibitors, DNAse inhibitors, etc. In one embodimentof the present invention, the sample is lysed directly in the formobtained from test-individuals. Proteinase inhibitors may e.g. compriseinhibitors of serine proteinases, inhibitors of cysteine proteinases,inhibitors of aspartic proteinases, inhibitors of metallo proteinases,inhibitors of acidic proteinases, inhibitors of alkaline proteinases orinhibitors of neutral proteinases. In certain embodiments of the presentinvention the inhibition of enzymes may be achieved by chemical meanssuch as e.g. denaturation of the enzymes by means of salt concentration,pH, chaotropic agents or the like.

In certain embodiments of the invention in order to obtain optimalresults of the assay, the pH of a sample medium that can be directlyapplied to the assay system is around neutral. In further embodimentsthe pH of the sample medium is within the range of 4 to 10. In certainother embodiments, the pH is in a range from 5 to 9. In a preferredembodiment, the pH is in a range from 6 to 8. In a more preferredembodiment, the pH is in the range from 6.5 to 7.5.

Examples of denaturing agents for sample media and/or for denaturing ofanalytes according to the present invention may for example be selectedfrom the substances given in Table 1.

TABLE 1 Sample medium Detergents: 0.1-1% SDS 0.2-3% SDS 0.2-3% DOC0.1-1% n-Octylglycoside 0.1-3% TRITON ® x-100% 0.1-1% Chaps 0.1-3%TWEEN ® Detergent-Mix: RIPA (1% NP40, 0.5% DOC, 0.1% SDS, PBS) 40-100%SOX (0.5% DOC, 0.5% n-Octylglycoside) 40-100% Special sample medium (3%TRITON ® X-100, 0.4% SDS, PBS) Commercial lysis media: Dynal (Dynal,Oslo, Norway) M-PER/B-PER (Pierce, Rockford, IL) Miscellaneous: 0.5-8 MUrea 1-99% Formamide GuaSCN 1-80% Formic Acid 1-70% Acetic Acid 1-50%Phosphoric acid Laemmli sample buffer (10-80% DMSO, 10-80% Formamide,50-70% formic acid, PBS, Citrate buffer pH 6.0, 500 mM NaCl in Phosphatebuffer)

All concentrations given in percent (%) throughout the whole disclosureof this invention refer to percent weight per volume (% w/v).

The sample media according to the present invention comprise inpreferred embodiments a denaturing agent. Such denaturing agent may beselected from a group comprising detergents or chaotropic agents.

In certain situations, the analytes may be degraded in the solubilizedsamples and may thus not be detected. This is particularly true, if thesamples are directly transferred to a lysing medium and stored thereinfor a certain period of time before further preparation. To preventdegradation, sample medium may furthermore comprise one or more agentsthat prevent the degradation of components within the raw samples. Suchcomponents may for example comprise enzyme inhibitors such as proteinaseinhibitors, RNAse inhibitors, DNAse inhibitors, etc. The inhibitors maye.g. comprise proteinase inhibitors selected from the compositions givenin Table 2.

TABLE 2 class of inhibited Inhibitor proteinase Aprotinin SerineBenzamidine Serine Bestatin Aminopeptidases Calpeptin Cysteine CystatinCysteine E-64 Cysteine EDTA Metallo Elastatinal Serine EST CysteineFetal calf serum all classes Leupeptin Serine/Cysteine a2-Macroglobulinall classes NCO-700 Cysteine Pefabloc = AEBSF Serine Pepstatin AAspartic PMSF Serine o-Phenanthroline Metallo

For stabilization purpose, the sample medium may also comprise bulkprotein (e.g. albumin such as bovine serum albumin or calf serumalbumin, soy lecithin or other bulk proteins) to compete in degradationwith the sample proteins. The bulk proteins may e.g. be present incombination with proteinase inhibitors or may be added instead ofproteinase inhibitors. In certain embodiments the sample medium may beselected to be compatible with the assay (e.g. ELISA) performance, sothat solubilized samples may directly be applied to the assay.

In certain embodiments the sample medium may also comprise substancesfor inhibition of microbial activity. Such substances may e.g. comprisemicrobizidic or microbiostatic substances. Such substances comprise e.g.sodium azide, proclin, 5-Bromo-5-Nitro-1,3-Dioxane,2-Methyl-4-Isothiazolin-3-One, 5-Chloro-2-Methyl-4-Isothiazolin-3-Oneand mercury containing compounds (e.g. thimerosal).

For overall stabilization of the analytes in the sample medium accordingto the present invention a heating step is applied.

A sample medium according to the present invention may in certainembodiments also comprise one or more denaturing agents. Suchcombinations may comprise the denaturing agents as disclosed herein inany suitable combination.

Heating as used in the context of the present invention shall refer to aprocess of subjecting the sample medium containing the solubilized bodysample to temperatures elevated relative to ambient temperature and maypertain to heating to any temperature below at or above the boilingpoint of a particular sample medium. Heating may e.g. comprisesubjecting to temperatures of 30 to 150° C. In certain embodimentsheating comprises subjecting to temperatures above 50° C. In a preferredembodiment of the invention heating shall refer to subjecting totemperatures between 70° C. and 130° C. In another preferred embodimentheating comprises subjecting to temperatures between 95 and 110° C.

In the context of the present invention heating is performed for aperiod of time that allows the analytes contained in the body sample tobe optimally stabilized in the sample medium. The period of timerequired for this may vary and is dependent on the type of samples. Ingeneral, the period of time is at least 3 minutes. The heating times(effective incubation times) at a given temperature may be chosen to bebetween 5 and 30 minutes. In a preferred embodiment of the invention theincubation time is between 10 and 27 minutes. In another preferredembodiment the incubation time is between 15 and 25 minutes. The overallheating time at a given temperature must be differentiated from the timefor incubation of the sample solution at a given temperature. Theheating time (effective incubation time) given above shall refer to thetime the sample solution is heated in a heating device comprising thetime period during which the temperature of the sample solution isequilibrated to the final incubation temperature. This is generallylonger than the period of time for incubation at the envisagedincubation temperature after the sample solution has been equilibratedto the incubation temperature. For mere incubation at the destinedtemperature a period of time from 1 minute to 30 minutes may be regardedsuitable for the methods according to the present invention. In apreferred embodiment of the present invention a period of time of 3 to15 minutes is used for heating. In certain embodiments of the inventionperiods of time between 5 and 12 Minutes are sufficient. The incubationtimes given are strongly dependent on the heating device used as well ason the vial type and on the sample media used. Therefore the time periodneeded for incubation may exceed or even be shorter than the time periodgiven above. Those of skill in the art know how to determine the periodof time necessary for incubation of an ambient temperature equilibratedsample solution that has been introduced into an already hot heatingdevice to allow for an incubation of the sample solution at theenvisaged incubation temperature for the planned period of time. Heatingdevices for performing the method of the present invention can be anyheating device applicable for heating a sample solution in a laboratoryfor a fixed period of time. Generally heating devices are equipped witha thermostat and may be set to hold a fixed pre-selected temperature.Such heating devices may e.g. comprise water baths, microwave ovens andheating blocks. In certain embodiments of the invention the heatingdevice is equipped with a timer that allows the user to control theincubation time easily. Otherwise the user may use an external timer(such as e.g. an alarm clock) to control the incubation time.

Stabilization as used in the context of the present invention shallrefer to a situation where an analyte that is incubated within thesample medium at a certain temperature degraded or otherwise modifiedthus rendering the analyte undetectable or detectable at an alteredlevel compared to the starting point of the incubation after a certainperiod of time. The stabilization shall in certain embodiments of theinvention refer to a stabilization of the detectable level of analyteswithin the sample solution. In certain embodiments a deviation of thelevel of analyte detectable after a certain period of time of 30% orless compared to the level detectable at the beginning of the timeperiod, is regarded as stable. In certain preferred embodiments adeviation of 20% or less is regarded as stable. In certain especiallypreferred embodiments a deviation of 15% or less is regarded asstabilization of the analyte.

In certain embodiments of the present invention the stabilization isespecially stabilization for storage and transport of the samples atambient temperature. Ambient temperature shall confer mainly totemperature between −20° C. and +50° C. Stabilization in specialembodiments of the present invention may also refer to stabilization ofthe analyte at special temperature ranges and may also refer tostabilization for storage and transport condition between 2° C. and 30°C. In yet another embodiment the temperature range may be between −80°C. and 20° C. In certain embodiments the stabilization shall especiallyrefer to a state, where the temperature of the samples is not controlledat all, so that any temperature present in the transport or storageenvironment may be accepted for the sample and stability in the sense ofno alteration of the level of the analyte detectable by a certaindetection method takes place. In some embodiments of the presentinvention the stabilization of the analyte may also be achieved for anytemperature of storage and transport.

Denaturing an analyte or denaturation of an analyte as used in thecontext of the present invention shall refer to any kind of altering theanalyte or alteration of the analyte respective the native statethereof. In certain embodiments of the invention such alterationrespective the native state may comprise any kind of disruption of thenative or naturally occurring secondary, tertiary or quaternarystructure of an analyte (protein).

One aspect of the present invention pertains to a denaturing bioassay. Adenaturing bioassay in the context of the present invention is animmunochemical assay that is designed to detect the presence or absenceand or the level of an analyte within a sample solution. Such bioassayis preferably an assay where the detection of the analyte is carried outin a liquid phase or using reagents fixed to a solid phase. In any casea denaturing bioassay according to the present invention ischaracterized in at least one step of the immunochemical reaction beingcarried out in the presence of denaturing agents. In contrast to thesituation in a denaturing bioassay according to the present invention,in SDS-PAGE only the gel electrophoresis is carried out under denaturingconditions and the subsequent immunochemical detection reaction iscarried out, after the denaturing agents have been washed away. Anexample of a denaturing bioassay according to the present invention ise.g. a sandwich ELISA where the antigen capturing step is carried outdirectly from the sample solution containing the analyte and thedenaturing agent. In such case the sample solution comprising adenaturing agent is contacted to a solid phase fixed antibody directedagainst a particular analyte. The capture of the analyte by the captureantibody is performed and the denaturing agent is washed away only afterthat step. The advantage of such method is, that conformational changespresent in the denatured analyte are upheld during the capture step andthus interaction of the probe or antibody may take place with thedenatured analyte. This may in certain cases lead to improved accuracyin detection of the analyte. Especially where linear epitopes of ananalyte have been employed for generation of probes or antibodies thedetection of such analytes by said antibodies may be improved if theimmunochemical reaction is carried out under denaturing conditions.

In the art however there was the prejudice that high concentration ofdenaturing agents interfere with the immunochemical reaction and thusantigen binding cannot take place in a proper way if there is e.g. SDSpresent in the incubation solution. The inventors now found that suchelevated concentrations of denaturing agents may be of positive impacton the performance of an immunochemical detection reaction under certaincircumstances as described above.

The present invention provides a method for detecting one or moreanalyte proteins by an immunoassay, comprising the step of carrying outan immunoassay in the presence of one or more denaturing agents in aconcentration to allow to uphold the denatured status of the one or moreanalyte proteins. For example, the method comprises the steps of i)bringing a sample comprising the one or more analyte proteins intocontact with the one or more denaturing agents, ii) heating said samplefor at least 3 minutes in the presence of the one or more denaturingagents to allow the protein to be denatured and iii) performing one ormore immunoassays for detection of the one or more analyte proteinsusing the sample of (ii) as a specimen for the test.

Denaturing immunoassays according to the present invention may comprisebut not be limited to for example immunoprecipitation or immunologicalassays, such as EIA, ELISA, RIA, ECLIA, LIA, lateral flow assays, flowthrough assays, immunochromatographic strips, latex agglutination assaysetc. Immunoassays for use as denaturing immunoassays in the inventionmay comprise competitive as well as non-competitive immunoassays. Incertain embodiments antibodies or probes fixed to solid phases may beemployed in the immunoassays. Solid phases may comprise variousembodiments of solid substances such as planar surfaces, particles(including micro-, nano-particles or even smaller particles). In certainembodiments, particles may be provided as spheres, beads, colloids, orthe like.

The fixation of reagents to the solid phase in a test kit or an in-vitrodiagnostic device may be carried out via direct fixation or via indirectfixation. Direct fixation may be carried out by covalent binding,non-covalent binding, association, or adsorption to surfaces. Indirectfixation may be carried out through binding of the antibody to agentswhich themselves are directly fixed to solid phases. Binding agents, forexample, include avidin, streptavidin, biotin, digioxingenin, antibodiesor the like.

The immunoassay may comprise one or more further reactions withdetecting agents either recognizing the analytes or preferablyrecognizing probes used to recognize the analytes. The detectionreaction further may comprise a reporter reaction indicating the levelof the analytes.

Detection systems may be e.g. chromogenic systems, luminescence systems(electroluminescence, bioluminescence, photoluminescence,radioluminescence, chemiluminescence, electrochemiluminescence),fluorescence based systems, conductivity based detection systems,radiation (light, UV, X-ray, gamma etc.), plasmon resonance (e.g.Surface Plasmon Resonance SPR) or any other known method.

The denaturing agents used for such denaturing bioassay may in certainembodiments of the invention be part of the sample medium into whichbody sample are introduced for storage and transport before analysis. Inother embodiments the denaturing agent is contacted to the analyteproteins in a sample just before the step of denaturation. Denaturingagents used for the denaturing bioassay as disclosed herein may forexample comprise chaotropic agents such as e.g. urea, GuaSCN, formamide,detergents such as anionic detergents (e.g. SDS, N-lauryl sarcosine,sodium deoxycholate, alkyl-aryl sulfonates, long chain (fatty) alcoholsulfates, olefine sulfates and sulfonates, alpha olefine sulfates andsulfonates, sulfated monoglycerides, sulfated ethers, sulphosuccinates,alkane sulfonates, phosphate esters, alkyl isethionates, sucroseesters), cationic detergents (e.g. cetyl trimethylammonium chloride),non-ionic detergents (e.g. TWEEN® 20, Nonidet P-40, TRITON® X-100,NP-40, Igepal CA-630, N-Octyl-Glucosid) or amphoteric detergents (e.gCHAPS, 3-Dodecyl-dimethylammonio-propane-1-sulfonate,Lauryldimethylamine oxide) and alkali hydroxides such as e.g. NaOH orKOH. In certain embodiments the denaturing agents may also compriseinorganic or organic acids as components such as formic acid, aceticacid, phosphoric acid etc.

According to the present invention elevated concentrations of detergentsmay be present during immunoassay. In certain embodiments of theinvention the immunoassays are tolerant against concentrations of SDSabove 0.1%, in a preferred embodiment the immunoassays are tolerantagainst SDS concentrations above 0.3%. In another preferred embodimentthe immunoassays are tolerant against concentrations of SDS above 1%. Incertain embodiments of the invention the immunoassay may be tolerantagainst concentration of TRITON® X 100 from below 0.1% up to at least3%.

A further aspect of the present invention is an automated heating devicecomprising a timer that is designed to perform a heating cycle for amethod according to the present invention. The heating device isdesigned to bring samples solubilized in a sample medium to a targettemperature and to maintain the samples at the target temperature for acertain amount of time.

In the most preferred embodiment of the invention automated shall meanthat the temperature setting is preset within the device and the usercannot and need not set the temperature for incubation. Furthermoreautomated shall mean that also the time for incubation is preset in thedevice. The user may only start the preset incubation cycle withoutbeing in the position to influence the preset parameters. Suchautomation is suited to reduce errors respective the incubationparameters.

In certain embodiments the heating device is designed to bring thesamples to and to maintain the temperature within said samples at atemperature of 70° C. or higher. In further embodiments the heatingdevice is designed to bring the samples to and to maintain thetemperature within said samples at a temperature of 90° C. to 110° C. Inthe most preferred embodiment the heating device is designed to bringthe samples to and to maintain the temperature within said samples at atemperature of 95° C. to 99° C. Due to measuring artefacts and/ormeasuring imprecision the temperatures given are to be understood as+/−2° C.

The heating device is designed to maintain the temperature within thesample for a time of at least 5 minutes. In another embodiment theheating device is designed to maintain said temperature within thesample for 7 to 30 Minutes. In the most preferred embodiment the heatingdevice is designed to maintain the temperature within the sample for aperiod of time of 10 minutes to 27 minutes.

In certain embodiments the heating device may be automated in a way toallow automatic detection of the end of the incubation period withautomatic shut off of the device. In further embodiment the device maybe equipped to give an acoustic, vibration or optic signal at the end ofthe incubation period.

In a preferred embodiment the heating device may be a heating block withdrills that may take up sample vials.

The examples provided below are indicated for illustration of thesubject matter of the invention and are not intended to limit the scopeof the invention. Several variations to the examples as provided may beperformed by those of skill in the art. The examples therefore provideonly a limited scope of embodiments of performance of the methods underthe presently detailed invention. Based on the comprehensive testingperformed by the inventors, we believe that the methods disclosed iseasily transferable to other analytes and other denaturing agentswithout altering the positive effects of the inventive methods asdetailed herein.

EXAMPLES Example 1 Stabilization of p16^(INK4a), Ep-Cam and GammaCatenin Proteins in Samples from the Uterine Cervix

The levels of p16^(INK4a) protein were determined in cervical specimensobtained with a standard cervical sampling device. 11 samples weresubjected to a heating step according to the invention disclosed herein,and were analysed for the level of p16^(INK4a) after storage for acertain period of time thereafter; further 7 samples were directly usedfor analysis of the samples for the level of p16^(INK4a) protein byELISA technique.

Sample Preparation and Stabilization

For the present examples patient sample were collected by gynecologistsusing an endocervical brush or a CERVEX-BRUSH COMBI® (Rovers MedicalDevices). After preparation of conventional PAP-smear specimens from theendocervical brush, the brush containing residual sample material wasinserted into a sample collection vial (e.g. PP Vial by Sarstedt)containing 5 ml PST-buffer (PST buffer: 1% TRITON® X100 0.3% SDS, andphosphate buffered saline; the phosphate buffered saline containedProclin300 as a preservative for stabilization of the buffer beforeuse). The handle of the brush was removed and the brush tip was leftwithin the sample collection vial. The sample collection vial withsample material and collecting brush was heat-treated within a period oftime of 2 hours after collection of the sample, for 15 min at 95° C. ina water bath, alternatively 25 min at 100° C. in heating block could beused for heat treatment step.

A heating block with thermostat and with drills of appropriate size forvials (NeoLab Cat. No 2-2503) was used. Sample was stored without anyfurther handling (no centrifugation, brush left in vial) at roomtemperature for up to 14 days until analysis of the sample by ELISA asfollows:

Performing the ELISA

p16^(INK4a) level and/or concentration was determined in duplicatemeasurements taking 100 μl of sample/determination. Levels ofp16^(INK4a) were measured at for example 1 days, 2 days, 5 days and 6days after start of storage of samples at room temperature. Each datapoint was a mean of 2 measurements (variation coefficients below 10%).

ELISA-plates coated with p16^(INK4a) specific antibody clone mtm E6H4were used for the ELISA detection of p16^(INK4a). 100 μl of the lysedcell sample was added to each well. For purpose of calibration of thetest, different concentrations of recombinant p16^(INK4a) protein (0pg/ml, 50 pg/ml, 100 pg/ml, 200 pg/ml, 400 pg/ml, 800 pg/ml) wereincluded in the test. Samples were incubated for 1 h at roomtemperature.

Thereafter 3 washing steps were performed using an automated ELISAwasher. Horse radish peroxydase (HRP) conjugated secondary antibodyclone mtm D7D7 specific for p16^(INK4a) protein was used for detectionin the sandwich ELISA system. 100 μl of D7D7-HRP solution was added toeach well and incubated for 1 h at room temperature. Thereafter 3washing steps were performed using an automated ELISA washer. 100 μl ofTMB-substrate was added to each well. The ELISA plates were incubated at25° C. for exactly 15 min in the dark. Then the reaction was stopped byaddition of 80 μl 2.5M H₂SO₄. Within 5 min after stopping the reaction,OD 450 nm was determined. After evaluation of the results, each samplereturned a value for the OD. Using a calibration curve based on thecalibration samples included in the assay the OD were transferred torelative Units/mL.

Ep-CAM and gCatenin were determined from analogous samples usingspecific sandwich-ELISA based on following antibody-pairs: HEA-125(German Cancer Research Center) and MK1-410 (BioVendor) for Ep-CAM, 10F8(mtm laboratories) and 4C12 (mtm laboratories) for gCatenin.

Results

The p16^(INK4a) levels measured in the cervical specimens was reportedeither in OD (all data for one sample were obtained using only one ELISAplate) or in Units/mL. The levels measured in a respective sample atdifferent points in time are shown in FIG. 1 for samples not treatedwith heat and in FIG. 2 for samples treated with heat.

The data shown in FIG. 1 demonstrate that there was a significant lossof p16^(INK4a) signal in the ELISA if a sample was stored at ambienttemperature over a period of several days. This indicates that there wasdegradation of the p16^(INK4a) analyte protein in the sample. In FIG. 2,the results show that there was no loss of signal over the whole periodof time that the samples were subjected to heat treatment. Therefore,the level of p16^(INK4a) from cervical specimen samples obtained afterheat treatment was stabilized during total storage time.

In order to determine if the level of p16^(INK4a) in specimen sampleswas affected by variation of the heat treatment procedure, 4 consecutivealiquots were taken from 14 different samples incubated as follows:

-   -   Aliquot 1 taken after 15 min at 100° C.    -   Aliquot 2 taken after 25 min at 100° C.    -   Aliquot 3 taken after 35 min at 100° C.    -   Aliquot 4 taken after further 10 min at 105° C. (total duration        of heat treatment was 45 min)

The p16^(INK4a) concentration was determined in duplicate from eachaliquot separately, using the p16-ELISA. Average concentration,including error bars of 4 determinations, as well as coefficient ofvariation between 4 determinations, are given in FIG. 3. 4 U/mL were setas a limit of detection in the present circumstances. The results ofthis experiment show that no difference in p16^(INK4a) levels wasvisible when samples were heat-treated for different lengths of time.This demonstrates that a wide range of time periods may be applied.Increased duration or even increased temperature for the heat treatmentof samples did not interfere with the analyte determination using thep16-ELISA.

Treating a pool of several patient samples for 10 min at 80° C., 90° C.or 99° C. showed that the stabilization was dependent upon thetemperature of heat treatment. An aliquot of the pooled samples was leftuntreated and used as a control. As shown in FIG. 4, the control wasunstable when not subjected to heat treatment and was stabilized upontreatment temperatures. The observed stabilization was the best when theheat treatment was performed at a temperature above 90° C.

The results of gCatenin levels in 3 patient samples not treated by heatand 7 patient samples treated by heat are shown in FIGS. 5 and 6,respectively. Our results show that gCatenin was stabilized within thePST-buffer upon heat treatment.

Analogous results of Ep-CAM levels in 6 patient samples not treated byheat and 7 patient samples treated by heat are shown in FIGS. 7 and 8,respectively. Our results indicate that Ep-CAM was also stabilizedwithin the PST-buffer upon heat treatment.

Determination of p16^(INK4a) was performed from aliquots taken from thesame patient samples as the determination of Ep-Cam and gCatenin. Thedetermination of p16^(INK4a) was performed using distinct ELISA platesfor each analyte. However, it is possible to generate ELISA plates wheredetermination of two or more analytes is performed on one single plate.

In summary, the results of the outlined experiments show that atemperature applied during heat treatment was well suited above 90° C.for at least 5 min under the given conditions. Furthermore, extension ofthe treatment duration up to 45 min did not alter the analytes.Furthermore, the results show that stability of p16, gCatenin and Ep-CAManalytes were achieved for storage at ambient temperature at least 6days. The observed stabilization was not protein specific, because itwas observed for 3 different proteins.

The results also demonstrate that all analytes tested in the experimentcould be detected in an immunoassay, wherein the first incubation stepwas performed in the presence of 0.3% SDS and 1% TRITON® X100.

It can be noted that the heat treatment procedure resulted in astandardization of the extraction procedure of p16^(INK4a) from cellspresent in a patient sample. Due to the presence of the brush, which wasleft in the vial during the heat treatment, an efficient lysis of thecells and a homogeneous solubilization of analytes was achieved by heattreatment. The risk of loosing material left in the brush was greatlyreduced.

Example 1a Denaturing Immunoassay for p16^(INK4a) Protein in Samplesfrom the Uterine Cervix

In a variation to the protocol shown above, the inventors performedvarious experiments with different heating times and differentcompositions of sample media. Cervical sampling device (CYTOBRUSH®)containing cervical swab was introduced into a 1.5 ml reaction tubecontaining 700 μl of lysis buffer (PBS with 3% TRITON® X100; 0.5% SDS).The shaft was cut and the closed tube was vortexed for 30 seconds atroom temperature. Thereafter, brush was removed. Reaction tube washeated for 30 min at 95° C. 100 μl of lysate was pipetted per well in a96 well plate coated with p16^(INK4a) capture antibody (clone E6H4).Detection was performed with biotinylated p16^(INK4a)-specific detectionantibody and Streptavidin-HRP conjugate. DAB was used as substrate. Theresults are displayed in FIG. 9. It can be seen that p16^(INK4a) wasdetected by the ELISA assay in the presence of denaturing agents (0.5%SDS and 3% TRITON® X100). Moreover, there was correlation between theelevated p16^(INK4a) values (indicative for p16^(INK4a) overexpression)and the cytologically confirmed presence of an abnormality of the tissuetested.

Example 2 Performance of Denaturing Immunoassays for p16^(INK4a) UsingELISA and LIA Technique

The levels of p16^(INK4a) protein are determined in cervical specimensthat are obtained with a standard cervical sampling device. 12 samplesare subjected to a heating step according to the invention disclosedherein, and are analysed for the level of p16^(INK4a) by ELISA and LIAtechnique in the presence of high detergent concentrations.

Sample preparation and stabilization and ELISA are performed as shown inExample 1.

Performing the LIA

Dynabeads M-280 are coated using the standard coating procedure fromDynal with the p16^(INK4a) specific antibody clone mtm E6H4 and storedin a concentration of 5 mg/ml in storage buffer. Antibody clone mtm D7D7specific for p16^(INK4a) is labelled with isoluminol by a standardprocedure. The assay is performed with the DIASORIN LIAISON® analyzer.200 μl sample of the lysed cell sample is added to 100 μl tracersolution(monoclonal anti-p16^(INK4a) antibody D7D7 labelled with isoluminol in10 mM PBS pH 7.4, 1% BSA) and 20 μl antibody-coated magnetic particles.After 10 min incubation at 37° C., the particles are separated with amagnet and washed for three times with LIAISON® Wash/System Liquid.After the third wash step, the particles are separated again and thesupernatant is discarded. The chemiluminescent signal is generated byinjection of two ready-to-use trigger solutions (LIAISON® Starter Kit).For purpose of calibration of the test, different concentrations ofrecombinant p16^(INK4a) protein (0 pg/ml, 50 pg/ml, 100 pg/ml, 200pg/ml, 400 pg/ml, 800 pg/ml) are included in the test.

Results

The p16^(INK4a) levels measured in the cervical specimens are reportedin Units/mL.

The data shown in FIG. 10 show only minor differences in the p16^(INK4a)levels measured by both assay types. The experiment thus proves thatELISA and LIA techniques may be used to detect the analyte in thepresence of the denaturing agent in a concentration (0.3% SDS) thatallows to uphold the denatured status of the analyte protein.

Inventors show in further experiments that ELISA assay as well as LIAassay are equally performed for detection of several analytes (includingp16^(INK4a), gCatenin, Ep-Cam, HPV E7, CA19-9, MCM-2 and CDC-6), whichmay be detected reproducibly in the presence of significantly higherconcentrations of denaturing agents, e.g., in the presence of 1% SDS.Alternatively a concentration of 3% TRITON® X100 is successfully used inan ELISA format for the detection of analytes. In these cases, theinventors observe an improvement of reproducibility of the resultsgenerated by analysis of samples in immunoassay when denaturing agentsare present during the assay compared to cases where denaturing agentsare omitted. This indicates that denaturing agents in the range ofconcentrations as detailed herein are suited to improve the performanceand especially the reproducibility of immunochemical detection methods.

Example 3 Detection of Cervical Intraepithelial Neoplasia in an LateralFlow Test Format

9 cervical swabs (conditions of lysis were similar to those ofExample 1) were subjected to conventional PAP testing and to lysis,

in subsequent lateral flow based detection of overexpression of cyclindependent kinase inhibitor p16^(INK4a) in solutions prepared from thecells contained in the swabs. The lateral flow testing was performed asfollows:

Cell Lysis

Cervical swab samples were transferred to sample collection vial (SCV)containing 5 ml of PST-buffer. The sample collection vial was incubatedin the water bath for 15 min at 95° C. The sample collection vial wascooled down to room temperature, and 4 ml of the supernatant weretransferred without centrifugation to a fresh tube.

Performing the Lateral Flow Assay Applying Capture Antibody to Membrane

Stock solutions of p16^(INK4a) specific antibody clone mtm E6H4 andrecombinant p16^(INK4a) protein were separately diluted in TBS(containing 1% bovine serum albumin) to give two ready-to-use spottingsolutions each with a final antibody concentration of 1 mg/ml or finalrecombinant p16^(INK4a) concentration of 10 μg/ml. The two ready-to-usesolutions were spotted onto different locations of nitrocellulosemembrane at 30 μl/30 cm.

Schleicher&Schuell wicks were attached to one end of the nitrocelluloseand the dipsticks were dried for 15 min at 37° C. Then they were allowedto equilibrate at room temperature and cut into 4 mm width dipsticks.

Incubation with Samples

2 μl of p16^(INK4a) specific antibody (clone mtm D7D7), conjugated tocolloidal gold (40 nm particle size; OD 44) was added to 100 μl of eachsample lysate (comprising 0.3% SDS and 1% TRITON®), mixed well andtransferred to a microtiter well. Dipstick, coated with clone E6H4 ascapture antibody line and recombinant p16^(INK4a) as positive controlline, was added to the well containing sample, soaked and run tocompletion. The signal was read whilst dipstick was still wet.

Results

The results are shown in FIG. 11. In our testing format, all 3 samplesclassified as PAP IVa by PAP staining of a corresponding cytologicalspecimen and therefore representing dysplastic cells, gave clearlyvisible bands in the area of spotted capture antibody. In contrast, nobands were detected for residual 6 samples, classified as PAP II-IIw byPAP staining, and therefore representing normal cells. Positive controllines indicated functional test components. Negative control (Co)without sample did not show nonspecific staining.

Example 4 Immunochemical Analysis of Protein Levels of MCM-5 and MCM-2in Liquid Based Cytology Samples from Urine

20 LBC samples of urine cells in CytoLyt™ are used for the presentexample. Protein analysis is performed in an ELISA format as given inExample 1 using pairs of commercially available antibodies directedagainst the respective analyte proteins. In both cases, monoclonalantibodies are used as capture antibodies coated to the ELISA plates andpolyclonal antibodies are used as detection antibodies. Experimentalprocedures are performed as given in there.

In this example, the body samples are analysed for the analytesimmediately after lysis and heating the samples.

It can be shown that MCM-5 as well as MCM-2 are easily detected inlysates from urine LBC samples after lysis of such samples in the samplemedium as disclosed in Example 1 and after heating of the samplesolutions. The results obtained by the immunochemical assay on MCM-2 andMCM-5 correspond well to the results obtained from immunocytochemicalanalysis of the samples for the respective proteins. In cytology,immuno-cytological staining for MCM-5 protein is used as aid inassessment of diagnosis.

The experiments above demonstrate that the methods disclosed herein aresuited for stabilization of a wide range of analytes for storage andtransportation and subsequent analysis by immunoassay without the needfor preservative additions. Furthermore the experiments demonstrate thatdenaturing immunoassays apply to a wide range of analytes and can helpto improve the immunochemical detection thereof.

The present invention facilitates easy and cost effective preservationof analytes obtained from a mammalian body for subsequent analysis byimmunochemical analysis. The invention provides a method that obviatesthe use of toxic and hazardous ingredients that may otherwise be usedfor preservation of the analytes in the samples. Furthermore, thepresent invention allows for increased accuracy and reproducibility ofdetermination of analytes in samples and thus increases reliability ofimmunochemical detection methods. Therefore, the invention contributesto improved immunochemical methods that contribute to test resultssupporting the assessment of diagnosis of diseases in humans.

1. A method for preparing and stabilizing one or more analytes insolutions for a denaturing immunoassay of body samples, comprising thesteps of: i) obtaining a solubilized mammalian body sample containingone or more analytes in a sample medium comprising a denaturing agent,wherein the one or more analytes are HPV associated markers; ii)subsequently heating said sample medium containing the one or moreanalytes to a temperature of 90-110±2° C. for a period of time of 3 to45 minutes; iii) stabilizing and storing the one or more heated analytesin said sample medium; and iv) subsequently performing a denaturingimmunoassay on said sample medium containing the one or more analytesand the denaturing agent of SDS.
 2. The method of claim 1, wherein thedenaturing agents are selected from the group consisting of SDS, DOC,n-octylglycoside, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycolsolution, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate,polyoxyethylene-sorbitan monolaurate, N-lauryl sarcosine, sodiumdeoxycholate, alkyl-aryl sulfonates, long chain fatty alcohol sulfates,olefine sulfates and sulfonates, alpha olefine sulfates and sulfonates,sulfated monoglycerides, sulfated ethers, sulphosuccinates, alkanesulfonates, phosphate esters, alkyl isethionates, sucrose esters,cationic detergents, cetyl trimethylammonium chloride,nonylphenylpolyethylene glycol, (octylphenoxy)polyethoxyethanol,amphoteric detergents, 3-dodecyl-dimethylammonio-propane-1-sulfonate,and lauryldimethylamine oxide.
 3. The method according to claim 1,wherein the temperature is between 95° C. and 99° C.
 4. The methodaccording to claim 1, wherein the period of time is at least 5 minutes.5. The method according claim 5, wherein the period of time is between 7minutes and 30 minutes.
 6. The method according to claim 6, wherein theperiod of time is between 10 minutes and 27 minutes.
 7. The method ofclaim 7, wherein the period of time is between 20 and 25 minutes.
 8. Themethod according to claim 1, wherein the heating step is performed in aheating device selected from the group consisting of a water bath, apressure cooker, a microwave oven, an UV heating device, and a heatingblock.
 9. The method according to claim 1, wherein the body sample isselected from the group consisting of secretions, swabs, lavages, bodyfluids, semen, cell- and tissue-samples, liquid based cytology samples,blood, smears, sputum, urine, stool, liquor cerebrospinalis, bile,gastrointestinal secretions, lymph, bone marrow, aspirates and biopsiesof organs.
 10. The method according to claim 1, wherein said HPVassociated markers are derived from HPV genes L1, L2, E1, E2, E4, E5, E6or E7.
 11. The method according to claim 1, wherein said storing iscarried out at 2-30° C.
 12. The method according to claim 1, whereinsaid storing is carried out for 1, 2, 5, or 6 days.
 13. The methodaccording to claim 1, wherein said storing is carried out for up to 14days.
 14. The method according to claim 1, wherein said sample medium of(iii) is stored without further handling until subsequently performingthe denaturing immunoassay.
 15. A method for detecting one or moreanalyte proteins by an immunoassay, comprising the step of carrying outan immunoassay in the presence of one or more denaturing agents in aconcentration to allow to uphold the denatured status of the one or moreanalyte proteins, wherein said analyte proteins are HPV associatedmarkers.
 16. The method according to claim 15, further comprising thestep of heating and denaturing the one or more analyte proteins in thepresence of the one or more denaturing agents.
 17. The method accordingto claim 15, comprising the steps of: i) bringing a sample comprisingthe one or more analyte proteins into contact with the one or moredenaturing agents; ii) heating said sample for at least 3 minutes in thepresence of the one or more denaturing agents to denature the one ormore analyte proteins; and iii) performing one or more immunoassays fordetection of the one or more analyte proteins using the sample of (ii)as a specimen for the immunoassays.
 18. The method according to claim15, wherein the immunoassays are selected from the group consisting ofimmunoprecipitation assays, immunological assays, EIA, ELISA, RIA,lateral flow assays, flow through assays, immunochromatographic strips,and latex agglutination assays.
 19. The method according to according toclaim 15, wherein the denaturing agents are selected from the groupconsisting of an organic acid, an inorganic acid, SDS, DOC,n-octylglycoside, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycolsolution, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate,polyoxyethylene-sorbitan monolaurate, urea, formamide, GuaSCN, formicAcid, acetic acid and phosphoric acid urea, N-lauryl sarcosine, sodiumdeoxycholate, alkyl-aryl sulfonates, long chain fatty alcohol sulfates,olefine sulfates and sulfonates, alpha olefine sulfates and sulfonates,sulfated monoglycerides, sulfated ethers, sulphosuccinates, alkanesulfonates, phosphate esters, alkyl isethionates, sucrose esters,cationic detergents, cetyl trimethylammonium chloride,nonylphenylpolyethylene glycol, (Octylphenoxy)polyethoxyethanol,amphoteric detergents, 3-dodecyl-dimethylammonio-propane-1-sulfonate,lauryldimethylamine oxide, alkali hydroxides, NaOH, and KOH.
 20. Themethod according to claim 15, wherein the one or more denaturing agentsare present in a concentration of 0.3% w/v or higher in the samplesolution.